Interactions of the KIR (human), Ly49 (mouse) and CD94/NKG2A inhibitory receptors with self MHC helps to prevent NK cells from attacking normal cells. When confronting cells that extinguish MHC class I, the absence of inhibition leads to NK activation and target cell lysis ("missing self recognition"). Each NK cell expresses a more or less random selection of the ~10 KIR or Ly49 receptors, which differ in their capacity to bind different MHC alleles. In the last funding period, we demonstrated that some NK cells in normal animals lack any receptors that bind self MHC. Despite the absence of inhibition, these NK cells are self tolerant, and indeed exhibit an "anergic" or hyporesponsive functional profile in which responses to most stimuli are blunted. We showed earlier that NK cells in mice that lack MHC class I altogether (e.g. b2m-/- knockouts) are similarly hyporesponsive and are self-tolerant despite the absence of inhibition by MHC class I. Recently, we and others demonstrated that NK cell responsiveness is in fact tunable across a broad range, such that NK cells with no, one or several receptors for self MHC are successively more responsive. We propose to test two new hypotheses that are predicated on preliminary data in the application. The first set of data show that when splenic NK cells are transferred from normal mice to class I deficient mice, or vice versa, their functional activity is "reset" based on the MHC environment of the host. Conversely, published data suggest that conditions associated with infections may prevent resetting to the hyporesponsive state or even reverse the hyporesponsive phenotype. These data provide a plausible explanation for why NK responses become blunted in tumor bearing patients. Our first specific aim is to test the hypothesis that NK cells reset responsiveness levels dependent on their MHC environment and inflammatory context. We propose to test whether and how the resetting process is modified by inflammatory cytokines or viral infections, whether inactivation of NK cells in tumor bearing mice reflects the resetting process and whether this can be reversed with the appropriate cytokines or infections. Additional preliminary data in the application show that amongst the most frequently upregulated genes in hyporesponsive NK cells are the Ly49 genes themselves, and that this is manifested as an increase in the number of different receptors expressed by each hyporesponsive NK cell as compared to each responsive NK cell. The second specific aim of the proposal is to test the hypothesis that tonic (ligand-independent) inhibitory signaling by Ly49 receptors is a component of NK hyporesponsiveness. We propose to test whether high level expression of nonself specific Ly49 receptors, or truncated Ly49 receptors lacking a ligand-binding domain, can impose hyporesponsiveness in vivo. Furthermore, we will test whether deletion of the expressed Ly49 genes reverses this effect, and the role of inhibitory signaling in this process. PUBLIC HEALTH RELEVANCE: This research addresses how the activity of natural killer (NK) cells, which provide first line defense against cancer and many infections, is "turned down" to prevent the cells from attacking normal cells, and "turned up" in the context of disease to protect the host. The results of our studies will help elucidate why the immune response sometimes fails or conversely becomes overactive, and will therefore help to guide the design of therapeutic agents that help correct related disorders.